2.1. Therapy with Combined Antimicrobial Agents
Combinations of antimicrobial agents have been widely described for treatment of bacterial infections. The simultaneous administration of more than one antimicrobial agent has been suggested for several purposes: (1) to prevent or minimize the emergence of resistant mutants; (2) for the enhancement of therapeutic activity in the treatment of specific infections (for example, penicillin and certain aminoglycosides are recommended for the treatment of certain infections with gram negative bacteria); (3) to provide optimal therapy in severe infections for which the causative agent has not been clearly established (e.g., in the treatment of mixed bacterial infections); (4) to lessen the toxicity of individual drugs by reducing the dose of each in combination.
Certain combinations of antimicrobial agents demonstrate enhanced antimicrobial activity and clinical effectiveness in the treatment of some infections (see Table I).
TABLE I ______________________________________ ANTIMICROBIAL AGENTS USED IN THE THERAPY OF INFECTIONS.sup.1 MICROORGANISM DISEASE DRUG COMBINATION ______________________________________ Streptococcus Endocarditis Penicillin G + viridans Bacteremia streptomycin Streptococcus Septicemia Ampicillin or agalactiae Meningitis penicillin G + an aminoglycoside Group B Endocarditis Penicillin G + Streptococcus Bacteremia an aminoglycoside faecalis (enterococcus) Corynebacterium Endocarditis Penicillin G + sp. an aminoglycoside (diphtheroids) Listeria Meningitis Ampicillin + monocytogenes Bacteremia an aminoglycoside Endocarditis Pseudomonas Pneumonia Carbenicillin or aeruginosa Bacteremia ticarcillin + gentamycin or tobramycin Klebsiella Pneumonia A cephalosporin + pneumoniae gentamycin or tobramycin Escherichia coli Urinary tract Ampicillin + infection gentamycin or trimethoprim- sulfamethoxazole Shigella Acute Trimethoprim- gastroenteritis sulfamethoxazole Yersinia Yersiniosis Trimethoprim- enterocolitica sulfamethoxazole Pneumocystis Pneumonia in Trimethoprim- carinii impaired sulfamethoxazole hosts Brucella Brucellosis A tetracycline + streptomycin Yersinia Plague A tetracycline + pestis streptomycin Pseudomonas Glanders A tetracycline + mallei streptomycin Pseudomonas Melioidosis A tetracycline + pseudomallei chloramphenicol Mycobacterium Pulmonary, Isoniazid + tuberculosis miliary renal, ethambutol or meningeal rifampin and other tuberculosis infections Mycobacterium Leprosy Dapsone + leprae rifampin Chlamydia Trachoma A sulfonamide + trachomatis a tetracycline Cryptococcus Meningitis Amphotericin B + neoformuns Flucytosine Gram negative Mecillinam + bacillary another .beta.-lactam infections ______________________________________ .sup.1 From Goodman & Gilman, 1980, The Pharmacological Basis of Therapeutics, Sixth Edition, pp. 1080-1105.
The antimicrobial activity of antibiotics used in combinations may result in a supra-additive (synergistic) effect. For example, in the treatment of bacterial infections combinations such as penicillin or ampicillin and streptomycin or gentamycin have been shown to have a supra-additive effect against enterococci infections. Similarly, carbenicillin or ticarcillin combined with an aminoglycoside such as gentamycin or tobramycin exhibit a synergistic effect in the treatment of Pseudomonas aeruginosa infection. Combined therapy using streptomycin together with tetracycline is more effective in the therapy of brucellosis than either agent alone, and a mixture of chloramphenicol plus a sulfonamide is more effective against meningitis due to Hemophilus influenzae.
The utility of combinations of drugs in the antimicrobial therapy of fungal infections has also been recognized. The concurrent administration of low doses of amphotericin B (20 mg daily) and flucytosine (150 mg/kg per day) for 6 weeks appears to be superior to using either drug alone in treating cryptococcosis as measured by a more rapid rate of sterilization of cerebrospinal fluid, reduced toxicity, and increased overall rate of cure. In addition, primary amebic meningoencephalitis has responded to a combination of miconazole, rifampin and intrathecal amphotericin B. Combinations of amphotericin B with other agents including flucytosine, rifampin, or tetracycline have been found to enhance antifungal activity.
Similarly the use of combinations of antiviral agents is currently being explored. Recently, the combination of acyclovir and vidarabine was reported to be more effective than the individual drugs in diminishing the development of clinical signs of herpes simplex virus type 1 infection in hairless mice (Park, et.al., 1984, The Journal of Infectious Diseases 149(5): 757-762).
However, combinations of drugs may be antagonistic rather this synergistic. For instance, the addition of a bacteriostatic drug (tetracycline) to a bactericidal drug (penicillin) produces a decrease in activity since penicillins can act only against microorganisms that are multiplying. Thus, a number of in vitro assays are used to predict the potential therapeutic efficacy of combinations of antibiotics. These assays quantitate the effects of the antibiotics on bacterial growth in vitro.
One method, which is used to predict the efficacy of antibacterial agents is described by Scribner et.al., (1982, Antimicrobial Agents and Chemotherapy 21(6):939-943) and in Goodman & Gilman (1980, The Pharmacological Basis of Therapeutics, Sixth Edition, pp. 1097-1098) and is referred to as the checkerboard assay. This assay involves serial two-fold dilutions of the antibiotics individually and in combination in broth which is then inoculated with the microorganism to be tested. After incubation, the minimum inhibitory concentration (MIC) of each drug used individually and in combination is determined (N.B., the MIC is the lowest concentration of the drug that inhibits growth in the medium). Synergism is indicated by a decrease in the MIC of each drug when used in combination. Antagonism is indicated by an increase in the MIC of either or both drugs when used in combination. This assay is described in more detail infra and is used in the present invention to determine whether certain combinations of antimicrobial agents (e.g., antibacterial or antifungal) are non-antagonistic.
Another method for the evaluation of drug combinations involves quantifying the rate of bacteriocidal action. Identical cultures are incubated with antibiotics added singly or in combination. Synergism is indicated if a combination of antibiotics is more rapidly bacteriocidal then either drug alone.
Similarly, combinations of antiviral agents may be assayed in vitro and classified as synergistic, additive, antagonistic, etc. Such an assay is described by Park et.al., 1984, The Journal of Infectious Diseases 149(5): 757-762. Briefly, the assay involves infection of confluent host cells in vitro with virus and treatment of the infected cells with various concentrations of the antiviral agents individually and in combination. After incubation, the virus titers recovered from the treated cells are determined by comparing the degree of titer reduction obtained when each drug is used singly to the degree of titer reduction obtained when the drugs are used in combination. This assay is also described in more detail infra and is used in the present invention to determine whether certain combinations of antiviral drugs are non-antagonistic.
In many instances, concurrent therapy with certain antimicrobial agents is further complicated because agents which exert a synergistic effect in vitro cannot be formulated in a single mixture for use in vivo. Mixtures of gentamycin and nafcillin at therapeutically effective concentrations result in the formation of complexes that precipitate out of solution and, therefore, are not administered in vivo. In fact, certain drug combinations are not recommended for use in vivo due to drug incompatibility (i.e., either inactivation of the drug or formation of a precipitate). For example, it is recommended that the following antibiotics not be mixed with any other drug: gentamycin, kanamycin, lincomycin, cephalothin, and ampicillin (Davis and Abbitt, 1977, JAVMA 170(2): 204-207). Finally, certain agents cannot be solubilized in the same media due to chemical restraints (e.g., a lipid soluble compound and a water soluble compound). These limitations reduce the possible combinations of agents that may be used to obtain enhancement of biological activity in combined therapy. For a review of the topic see Goodman and Gilman, 1980, The Pharmacological Basis of Therapeutics Sixth Edition, pp. 1080-1106 and 1239-1240 and Davis et.al., 1980, Microbiology, pp. 574-583.